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Taurine, which has multiple important physiological roles in teleost fish and mammals, is an amino acid not found in alternative protein sources not derived from animals. Although taurine is found in fish-meal-based feeds, its high water solubility leads to lower taurine levels in reduction-process-based feeds, which marine carnivores such as Cobia Rachycentron canadumare adapted to in their natural diets. Graded taurine supplementation (0, 0.5, 1.5, and 5.0%) added to a traditional fish-meal-based formulation was examined in two growth trials with Cobia: one initiated with 10-g individuals and the second initiated with 120-g individuals. During the first trial, in which growth as weight gain ranged from 123 to 139 g per fish, there was an increase in dietary taurine and a decrease in the feed conversion ratio from 1.04 to 0.99. During the second trial, in which growth ranged from 227 to 313 g gained per fish, there was no significant difference in performance characteristics between dietary treatments. Messenger RNA transcript expression levels for two of the genes involved in taurine synthesis, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), as well as the membrane-bound taurine transporter, TauT, were also measured at the conclusion of the second trial. Increasing dietary taurine in a diet containing 34.5% fish meal did not result in significantly different growth or production characteristics in Cobia, but did result in significantly increased taurine levels in fillet, liver, and plasma.

The karlotoxins are a family of amphidinol-like compounds that play roles in avoiding predation and in prey capture for the toxic dinoflagellate Karlodinium veneficum. The first member of the toxin group to be reported was KmTx 1 (1), and here we report an additional five new members of this family (3−7) from the same strain. Of these additional compounds, KmTx 3 (3) differs from KmTx 1 (1) in having one less methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 (4) and 10-O-sulfo-KmTx 3 (5) were identified. Likewise, 65-E-chloro-KmTx 1 (6) and 10-O-sulfo-KmTx 1 (7) were also isolated. Comparison of the hemolytic activities of the newly isolated compounds to that of KmTx 1 shows that potency correlates positively with the length of the lipophilic arm and is disrupted by sulfonation of the polyol arm.

Karlodinium micrum(family Dinophyceae) produces toxic compounds (KmTx's) that are associated with fish kills. For zebrafish Danio reriolarvae (24 h old) exposed to either KmTx 1 or KmTx 2, mortality (100% in 24 h) was observed at toxin concentrations of 1 µg/mL or more, whereas no mortality occurred after 24 h at concentrations of 0.5 µg/mL or less. Zebrafish and sheepshead minnow Cyprinodon variegatusjuveniles (60–90 d old) exposed to KmTx 2 were more sensitive to the toxin's effects than larvae were; mortalities in the juveniles began at 0.1–0.5 µg/mL. In whole, sectioned juvenile zebrafish, gills were the primary site showing injury by light microscopy. Histology of gills in both species treated with 0.5 µg KmTx 2/mL (100% mortality in 1 h) showed epithelial necrosis and shortening or loss of secondary lamellae. Histology of zebrafish gills treated with 0.05 and 0.1 µg/mL (0–44% mortality in 4 h) showed clubbing and bridging between secondary lamellae within 4 h of exposure. Sheepshead minnow exposed to 0.1 µg/mL showed similar gill pathology but no mortality after 6 h. Sheepshead minnow exposed to KmTx 2 at 0.5 µg/mL or more all died in less than 1 h. Transmission electron microscopy of gills of moribund zebrafish exposed to 0.1 µg/mL revealed extensive cellular hypertrophy and lysis of epithelial and chloride cells. Because concentrations of KmTx 1 and KmTx 2 range from 0.1 to 0.8 µg/mL in filtered water samples from K. micrum–associated fish kills, these results suggest that the concentrations of KmTx 1 and KmTx 2 found during fish kills are acutely toxic to fish and that gills are a primary target.

Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 μl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 ± 1.6 pmol mg−1h−1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg−1h−1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.

Plasma triacylglycerol, phospholipid, cholesterol, cholesterol esters, fatty acids, and total lipids were measured in 30 female and 20 male desert tortoises (Gopherus agassizii) during the annual reproductive cycle in the eastern Mojave desert, Nevada. Blood samples were collected at monthly intervals from April to October. All lipid fractions, with the exception of free fatty acids, were significantly higher in female plasma than in male plasma in all months of the year. In contrast, free fatty acids were higher in male plasma than in female plasma in all months. The seasonal pattern in estradiol secretion mirrored that of triacylglycerol, phospholipid, cholesterol, and total lipid, all of which showed a significant correlation with the hormone. Estradiol and the vitellogenesis-associated lipids were all significantly higher in August, September, October, and April than in June. The seasonal variation in cholesterol ester levels in females did not correlate with any of the reproductive events and did not appear to be involved in yolk precursor formation. Total lipid in males showed a negative correlation with testosterone and spermatogenesis. Individual fatty acids in the June and August samples (at the highest and lowest estradiol levels) were compared in male and female plasma. The percent of C18:3n3, C18:2n6, C18:1n9, C20:5n3, and C22:5 were significantly higher in the June female plasma sample than in the August sample. Docosahexanoic (C22:6n3) acid was barely detectable in female plasma in either month. J. Exp. Zool. 293:703–711, 2002. © 2002 Wiley-Liss, Inc.

The formation of estrogens from androgens in all vertebrates is catalyzed by the “aromatase” complex, which consists of a membrane bound P450 enzyme, P450 aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5°C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5°C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5°C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5°C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5°), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important. J. Exp. Zool. 290:673–690, 2001. © 2001 Wiley-Liss, Inc.

This report describes the structure of the 5′-flanking regions of both the CYP19A1 and A2 genes that were isolated from the genome of the zebrafish (Danio rerio). Consensus sequences of three cAMP-responsive elements (CRE), an aryl hydrocarbon-responsive element (AhR/Arnt), a steroidogenic factor 1 (SF-1) site, and a TATA box were observed in the 5′-flanking region of CYP19A1. In contrast, the 5′-flanking region of CYP19A2 was located upstream of an untranslated exon and possessed consensus sequences of a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor α/retinoid X receptor α heterodimer-responsive element (PPARα/RXRα), and a TATA box. Primer extension analysis revealed that the predominant transcription initiation sites for CYP19A1 and A2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively. These analyses indicate that substantially different regulators, including a variety of environmental xenobiotics, control the expression the two CYP19 genes.

Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17α-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the “extra-gonadal” form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475–483, 2001. © 2001 Wiley-Liss, Inc.

In all species of crocodilians, sex is determined not by genetic mechanisms, but by the temperature at which the egg is incubated. In the American alligator (Alligator mississippiensis) the thermosensitive period (TSP) for sex determination is a 7- to 10-day window within stages 21–24 of development, around the middle third of the incubation period. Treating embryos with estrogen during the TSP produces female offspring, even at male incubation temperatures. Conversely, blocking embryonic estrogen synthesis at female-inducing temperature prevents development of the female phenotype. Therefore, it has been suggested that estrogen plays a role in determination of sex in the alligator. Estrogen is produced from an androgen substrate by cytochrome P450 aromatase (CYP19). If estrogen plays a critical role in sex determination, there should be differences in aromatase expression between embryos at male- and female-producing temperatures during the TSP. Therefore, to address this question, we cloned and characterized the alligator CYP19 cDNA. Based on the sequence information, a quantitative kinetic reverse transcriptase–polymerase chain reaction (TaqMan) assay was designed to measure expression of the alligator aromatase gene in RNA extracted from the gonadal and brain regions of alligator embryos incubated at male- or female-producing temperatures from prior to the TSP through hatching. Aromatase expression was detected in the brain region from the earliest stage tested (stage 20) through hatching. The hypothalamus had significantly higher expression than the forebrain or hindbrain in both male and female embryos. Expression was not significantly different in the gonadal region between embryos at male and female temperatures until after the TSP, when there was a dramatic increase in expression at female temperature. These data indicate that aromatase expression and, thus, estrogen production, are not the initial trigger for sex determination but play an essential role in ovarian differentiation in the alligator. J. Exp. Zool. 290:439–448, 2001. © 2001 Wiley-Liss, Inc.

The effects of neurotensin on pancreatic exocrine secretion were examined in fasted, conscious White Leghorn hens. A cannula was surgically implanted in the central duct serving the ventral lobe of the pancreas in order to collect pure pancreatic juice. Following recovery, neurotensin was infused intravenously at 3.6 or 10.8 pmol/kg*min. The volume and pH of the pancreatic secretions were recorded and total pancreatic protein concentration, amylase, lipase, trypsin, and chymotrypsin activity were measured every 30 min for 2 hr and compared to secretions following the infusion of 0.9% saline. Our results demonstrated that neurotensin did not affect the pH nor the pancreatic juice protein concentration, but did increase secretion rate following neurotensin infusion at 3.6 pmol/kg*min. Amylase activity was significantly depressed during neurotensin infusions, while lipase (both pancreatic and carboxylester lipase) activity was significantly elevated. The ratio of amylase to lipase activity was especially depressed by neurotensin infusion at 10.8 pmol/kg*min. Insufficient secretory activity prevented a balanced statistical analysis of chymotrypsin activity, but from a pooled analysis, neurotensin had no effect on protease activity in the pancreatic juice. These results support our current research indicating that neurotensin may be a hormonal regulator of postprandial lipid digestion in chickens. J. Exp. Zool. 283:455–462, 1999. © 1999 Wiley-Liss, Inc.

Thrushes consume a variety of fruit that differ greatly in nutrient composition, especially lipid content. Thrushes may be efficient at extracting energy from fruit diets by changing how they digest meals differing in lipid content. We tested for this capability in American robins (Turdus migratorius) by acclimating them to diets differing in lipid content and measuring passage rate and absorption efficiency of ingested lipids with radiolabeled markers. Birds acclimated to high-lipid (26% and 40% dwt) diets ingested less dry matter and retained ingested lipids in their gastrointestinal tract three times longer than when acclimated to a low-lipid (9%) diet. Absorption efficiency did not differ among diets (˜74%), suggesting robins minimize feeding time rather than maximize rate of energy gain when processing meals. Thus, robins slowed intake and passage rate of dietary lipids with increasing dietary lipid concentration while maintaining a steady lipid absorption efficiency. J. Exp. Zool. 283:408–417, 1999. © 1999 Wiley-Liss, Inc.

Alcohol dehydrogenases (ADH) from the F1progeny of all pairwise crosses between 12 null-activity mutants and crosses between these mutants and four active variants, ADHn5ADHF, ADHDand ADHS, were analyzed for the presence of active or inactive heterodimers. Gels were stained for ADH enzyme activity, and protein blots of duplicate gels were probed with ADH-specific antibody to detect cross-reacting material. Crosses between the three major electrophoretic variants. ADHF, ADHSand ADHD, all produced active heterodimers. Four mutant proteins (ADHn2, ADHn4, ADHn10and ADHn13) did not form heterodimers with any other ADH subunit tested. Of the 28 crosses involving the remaining null activity mutants, 22 produce heterodimers. Twelve of these exhibit partial restoration of enzyme activity. In five cases of active heterodimers from null-activity crosses, Adhn11supplied one of the subunits. In two crosses involving the active variant ADHD, the null activity mutant subunits (ADHn8and ADHn3) destabilized the heterodimer sufficiently to cause inactivation of the ADHDsubunit. In the cross between AdhFand Adhn3, the activity of the ADHFsubunit was also greatly reduced in association with the ADHn3subunit. Two crosses (Adhn1× Adhn11and Adhn5× Adhn12) result in partial restoration of one of the homodimeric proteins (ADHn1and ADHn12, respectively), as well as forming active heterodimers.

Using protein blotting and an immuno-overlay procedure, we have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutants have retained the ability to form dimers under native conditions. None of the inactive mutant proteins has the ability to form the "adduct-bound" isozyme. We have found no correlation between protein pI and in vivostability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

Two formaldehyde-induced mutations at the DrosophilaAdh locus (Adhfn45 andAdhfn46) were analyzed by determining RNA structures at different developmental stages, polymerase chain reaction (PCR) amplification of the affected genomic regions, and direct sequencing of the resulting double-stranded DNA fragments.Adhfn46 adults and larvae accumulate abundant ADH-like distal (adult) and proximal (larval) transcripts that are shorter than transcripts in wild-type flies by a lesion located in the second ADH protein-coding exon. Direct sequencing of the amplified DNA region showed thatAdhfn46 contains a 69-bp in-frame deletion that removes 23 amino acids near one border of the second exon. Consistent with these findings, we observed a shorter ADHfn46 protein present at only 3% of wild-type levels. In contrast,Adhfn45 adults and larvae accumulate much smaller amounts of ADH-like distal and proximal transcripts. Both RNAs have an identical aberration in RNA splicing of the 65-base intron sequence. Direct sequencing of the amplified mutated DNA region showed thatAdhfn45 contains a 21-bp deletion that removed and rearranged DNA at the 5' splice junction of the 65-bp intron. No ADH cross-reacting material is detected inAdhfn45 flies. Direct-repeat sequences (3–11 bp) are present flanking and within the mutated DNA regions. The patterns of DNA deletion and deletion accompanied by sequence addition at the mutant sites suggest a slipped mispairing mechanism during DNA replication or repair that involves local DNA homology.

Adhfn23 andAdhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene tht codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) ofDrosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis inAdhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast,Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As withAdhfn23, the deletion inAdhfn24 results in a change in the reading frame. UnlikeAdhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on theDrosophila ADH mRNA levels and ADH protein levels are discussed.

Natural populations of Fundulus heteroclitus show extensive spatial variation in gene frequencies at four unlinked polymorphic loci. Large clinal changes in gene frequencies were found for Ldh-B, Mdh-A, and Gpi-B, whereas the spatial variation for the Pgm-B locus was small. Since the geographical area over which these clines are found is characterized by a steep thermal gradient, the clines in gene frequency are correlated with a directional change in mean water temperature. Maximum gene diversity of these four loci was correlated with annual fluctuations in water temperature. Temporal stability of the allelic frequencies was established for a 2–4 year period.

Electrophoretic analysis has shown populations of F. heteroclitus to possess variants at four enzyme-coding loci: Ldh-B, Mdh-A, Gpi-B, and Pgm-A. Based on the phenotypic distribution in the F1 generation, each variant segregates as an autosomally inherited codominant allele. A pairwise comparison of the expected phenotypic classes among these loci showed no evidence of strong linkage; however, weak linkage could not be ruled out. Despite the considerable genetic divergence of populations from the geographical extremes of this species, offspring resulting from crosses between individuals from these localities show viabilities similar to those found for crosses of local populations.

Sex determination in egg-laying amniotes may be fundamentally different from that of placental mammals. The mammalian ovary differentiates normally in the absence of estrogen, whereas estrogen seems to be crucial for proper ovarian development in birds, reptiles, and lower vertebrates. Estrogens are produced normally by the biosynthetic conversion of androgens by the enzyme aromatase (CYP19), which is the sole mediator of this reaction. Aromatase inhibitors are capable of reversing females to males in turtles and chickens; therefore, a role for aromatase as the female sex determinant has been postulated for species in which sex determination is temperature-dependent. The entire aromatase coding sequence (1,509 base pairs) from adult terrapin ovaries was cloned, and Northern analysis indicates a single transcript (2.4 kb) for adult ovaries, whereas male and female brains express a 2.4-kb as well as a 9.6-kb transcript. Using a sensitive (attomole sensitivity) competitive RT-PCR technique, aromatase transcript abundance was quantified during embryonic development for embryos treated with and without estrogen. Aromatase is transcribed, well before the temperature-sensitive, (stage 12), at both male and female temperatures in the brain. There is a switch to lower aromatase transcript abundance in the female brain concurrent with an exponential rise of aromatase transcript in the putative ovary. Transcripts remain below the detection limits in the putative testes but exhibit female levels of aromatase transcript when treated with estrogen. Aromatase mRNA levels are generally reduced in the brain by estradiol application. On the basis of these findings, we have postulated a model based on the competition between 5α-reductase and P450 aromatase for androgen substrate in both the brain and the undifferentiated gonad to explain the TSD phenomenon in reptiles. J. Exp. Zool. 281:428–449, 1998. © 1998 Wiley-Liss, Inc.

ERYTHROCYTE organic phosphate concentration is known to be an heritable trait in both humans and rats1–3. In humans this trait has been correlated with genetic variability of both pyruvate kinase and hexokinase2. However, similar phenomena have not been reported in lower vertebrates. During our studies on the allosteric regulation of fish haemoglobins by ATP (ref. 4) we observed significant variability in red cell ATP concentrations among individuals of the killifish Fundulus heteroclitus, and wondered whether this variability in organic phosphate could be the result of a genetic component. Consequently, we analysed F. heteroclitus for red cell ATP, concomitantly screening for enzyme variants. We report here that not only are ATP levels under genetic control, but they are also highly correlated with lactate dehydrogenase (LDH) phenotype. To our knowledge, this is the first such correlation reported. Moreover, as ATP is the major allosteric modifier of F. heteroclitus haemoglobin–oxygen affinity5,6, this study provides a basis for an association between LDH and the fish's external environment through the physiological role of haemoglobin at the organism–environment interface.

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Two formaldehyde-induce, homozygous viable ADH-negative mutants, Adhfn4and Adhfn6, possess no material that cross-reacts with antibody directed gainst ADH, no mature mRNA of wild-type size, and greatly reduced amounts of RNA that hybridizes with an Adh probe. We have cloned the genomic DNA sequences from these mutants in bacteriophage λ Charon 4 and subcloned the Adh region into plasmid vector pBR327. Restriction analyses revealed one small deletion in each of these mutants and DNA sequencing showed that the splice junctions of he 65-base pair (bp) intervening sequence (IVS) were altered. Both cloned mutant Adh genes, as well as the wild-type gene, are capable of promoting correct specific transcription, initiation in HeLa cell nuclear extracts in vitro. We conclude that Adhfn4and Adhfn6 are defective in RNA processing. Our results provide evidence for the importance of the splice junction sequences in normal ADH RNA processing and stabilization in Drosophila. We also speculate that splicing of ADH RNA proceeds in a nonrandom manner: mutations in one of the intervening sequences appear to cause accumulation of a large ADH RNA containing at least one other IVS